Neuroinflammation has been recognized as an essential player in the pathogenesis of Alzheimer's disease (AD), especially for the late-onset AD. This notion is supported by the facts that glial activation and elevated cytokines have been observed in AD animal models and patients. Furthermore, genome-wide associated studies have identified inflammatory genes in the innate immune system, such as CLU, CR1 and TREM2, as AD risk factors. Recently, the NLRP3 inflammasome, a multiprotein platform that tightly regulates the innate immune response, has been suggested to play critical roles in AD development. Activation of the NLRP3 inflammasome is responsible for the production of pro-inflammatory interleukin (IL)-1? and IL-18, ultimately leading to inflammatory responses. Given the important role of the NLRP3 inflammasome and IL-1? in AD, development of selective NLRP3 inflammasome inhibitors (NLRP3Is) as chemical probes with well- defined mode of action will not only enhance our current knowledge on the NLRP3 inflammasome in AD pathogenesis, but also provide translational promise to this disease. Recently, we developed a lead inhibitor that blocks the assembly and activation of the NLRP3 inflammasome, resulting in inhibition of IL-1? production both in vitro and in vivo. The central hypothesis of this proposal is that the NLRP3 inflammasome is involved in chronic inflammatory responses of AD, and pharmacological suppression with small molecule inhibitors that directly target the NLRP3 inflammasome platform will prevent or inhibit AD disease progression. In support of this hypothesis, our preliminary studies showed that the lead inhibitor engaged the NLRP3 inflammasome, reduced AD pathology, and improved cognitive functions in transgenic AD mouse models, thus providing proof-of-concept for developing NLRP3Is as in vivo probes. Furthermore, our preliminary structure activity relationship (SAR) studies confirmed that this chemical scaffold can be optimized to improve inhibitory potency and pharmacokinetic properties. The goal of this proposal is to understand the chemical space of this scaffold and to develop more potent analogs by comprehensive SAR studies as in vivo probes and three specific aims are proposed to achieve our objective in this application. In Aim 1, new analogs of this lead structure will be designed and synthesized to provide understanding of SAR for this scaffold that will guide the development of more potent inhibitors. In Aim 2, the designed new analogs will be evaluated in tiered biological systems for potency, selectivity, target engagement, and immunotoxicity. In aim 3, the top candidate inhibitor identified from Aim 2 will be tested to confirm the in vivo efficacy in a transgenic AD mouse model. The proposed research is highly significant because successful development of novel and selective NLRP3Is will not only provide effective pharmacological tools to precisely define the contribution of the NLRP3 inflammasome in disease pathogenesis, but also provide promising candidates for clinical studies, thus offering translational potential to achieve clinical benefits.